Chemical ligation of nucleic acids

ABSTRACT

The invention relates to the field of nucleic acid analysis. More particularly, the invention relates to compositions and methods used for the detection of sequence variations or single nucleotide polymorphisms (SNPs) in a nucleic acid of interest.

FIELD OF THE INVENTION

The invention relates to the field of nucleic acid analysis. Moreparticularly, the invention relates to compositions and methods used forthe detection of sequence variations or single nucleotide polymorphisms(SNPs) in a nucleic acid of interest.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal and mutantgenes and identifying mutant genes such as oncogenes, in typing tissuefor compatibility preceding tissue transplantation, in matching tissueor blood samples for forensic medicine, and for exploring homology amonggenes from different species.

Gene probe assays are commonly used to analyze the relationship betweengenetic variation and phenotype by identifying polymorphic DNA markers,such as single nucleotide polymorphisms (SNPs). Some SNPs, particularlythose in and around coding sequences, are likely to be the direct causeof therapeutically relevant phenotypic variants and/or diseasepredisposition. There are a number of well known polymorphisms thatcause clinically important phenotypes; for example, the apoE2/3/4variants are associated with different relative risk of Alzheimer's andother diseases (see Cordor et al., Science 261(1993).

Ideally, a gene probe assay for the detection of polymorphisms should besensitive, specific and easily automatable (for a review, see Nickerson,Current Opinion in Biotechnology 4:48-51 (1993)). The requirement forsensitivity (i.e. low detection limits) has been greatly alleviated bythe development of the polymerase chain reaction (PCR) and otheramplification technologies which allow researchers to amplifyexponentially a specific nucleic acid sequence before analysis (for areview, see Abramson et al., Current Opinion in Biotechnology, 4:41-47(1993)). For example, multiplex PCR amplification of SNP loci withsubsequent hybridization to oligonucleotide arrays has been shown to bean accurate and reliable method of simultaneously genotyping hundreds ofSNPs; see Wang et al., Science, 280:1077 (1998); see also Schafer etal., Nature Biotechnology 16:33-39 (1998).

Specificity, in contrast, remains a problem in many currently availableassays gene probe assays. The extent of molecular complementaritybetween probe and target defines the specificity of the interaction.Variations in probe composition, the concentrations of probes, oftargets and of salts in the hybridization medium, in the reactiontemperature, and in the length of the probe may alter or influence thespecificity of the probe/target interaction.

It may be possible under some circumstances to distinguish targets withperfect complementarity from targets with mismatches, although this isgenerally very difficult using traditional technology, since smallvariations in the reaction conditions will alter the hybridization. Newexperimental techniques with the necessary specificity for mismatchdetection with standard probes include probe digestion assays in whichmismatches create sites for probe cleavage and DNA ligation assays wheresingle point mismatches prevent ligation.

There are a variety of enzymatic and non-enzymatic methods available fordetecting sequence variations. Examples of enzyme based methods todetect variations in nucleotide sequences include, but are not limitedto, Invader™, oligonucleotide ligation assay (OLA) single base extensionmethods, allelic PCR, and competitive probe analysis (e.g. competitivesequencing by hybridization).

A number of non enzymatic or template mediated chemical ligation methodshave been developed that can be used to detect sequence variations.These include chemical ligation methods that utilize coupling reagents,such as N-cyanoimidazole, cyanogen bromide, and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. SeeMetelev, V. G., et al., (1999) Nucleosides & Nucleotides, 18:2711;Luebke, K. J., and Dervan, P. B. (1989) J. Am. Chem. Soc., 111:8733; andShabarova, Z. A., et al., Nucleic Acids Research, 19:4247. Otherchemical ligation methods react a 5′-tosylate or 5′-iodo group with a3′-phosphorothioate group, resulting in a DNA structure with a sulfurreplacing one of the bridging phosphodiester oxygen atoms. See Gryanov,S. M., and Letsinger, R. L., (1993) Nucleic Acids Research, 21:1403; Xu,Y. and Kool, E. T. (1997) Tetrahedron Letters, 38:5595; and Xu, Y. andKool, E. T., (1999) Nucleic Acids Research, 27:875. Some of theadvantages of using non-enzymatic approaches for the detection ofpolymorphisms include lower sensitivity to non-natural DNA analogstructures, ability to use RNA target sequences, lower cost and greaterrobustness under varied conditions.

PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, PCTUS99/01705, WO96/40712 and WO98/20162, all of which are expresslyincorporated by reference, describe novel compositions comprisingnucleic acids containing electron transfer moieties, includingelectrodes, which allow for novel detection methods of nucleic acidhybridization.

Accordingly, it is an object of the present invention to providenon-enzymatic methods in combination with electrochemical detection fordetecting single nucleotide polymorphisms in nucleic acid sequences ofinterest.

SUMMARY OF THE INVENTION

In accordance with the objectives outlined above, the present inventionprovides compositions and methods for detecting sequence variations innucleic acid sequences of interest. The sequence variation may include asingle base, i.e., a single nucleotide polymorphism, or several bases.The compositions and methods employ sequence-specific probes that permitthe use of non-enzymatic methods of chemical ligation for the detectionof sequence variations in a nucleic acid of interest. By using ligationprobes that are labeled at either the 5′ end with an iodide moiety or atthe 3′ end with a sulfur moiety, and that are either complementary overtheir entire length or differ in at least one base, nucleic acidscomprising sequence variations can be detected based on whether or not aligation product is formed. Additionally, one or more of the probes cancomprise electron transfer moieties (ETMs), the presence of which isused as an indication of the presence of a nucleic acid comprising asequence variation of interest.

The compositions of the present invention can further comprise a targetnucleic acid comprising a first domain capable of binding in asequence-specific manner to a first ligation probe, a second domaincapable of binding in a sequence-specific manner to a second ligationprobe, one of which can comprise at least one electron transfer moiety(ETM), and a detection position for which sequence information isdesired. Either the first or second ligation probe includes aninterrogation position having a base capable of binding in asequence-specific manner to the base or bases located at the detectionposition on the target nucleic acid strand.

Preferably, the compositions of the present invention comprise one, two,three, and/or four ligation probes comprising interrogation positionsand at least one electron transfer moiety with a distinguishable redoxpotential. For example, one of the four ligation probes may include afirst base and an ETM with a first redox potential, the second ligationprobe may include a second base and an ETM with a second redoxpotential, the third ligation probe may include a third base and an ETMwith a third redox potential, and the fourth ligation probe may includea fourth base and an ETM with a fourth redox potential. Although anynumber of ETMs may be used in the compositions of the present invention,in preferred embodiments, the ETMs are ferrocene or ferrocenederivatives having redox potentials that are readily distinguishableusing the methods described herein.

According to the invention, a target nucleic acid comprising a firstdomain capable of binding in a sequence-specific manner to a firstligation probe, a second domain capable of binding in asequence-specific manner to a second ligation probe, and a detectionposition is contacted with a first ligation probe, and at least a secondligation probe comprising an interrogation position and an ETM with adistinguishable redox potential. If the base present at theinterrogation position is capable of pairing in a sequence-specificmanner with the base present at the detection position, a ligationcomplex comprising the target strand and a ligated strand comprising thefirst and second ligation probe is formed.

The identity of the base located at the interrogation position, andthus, the base present at the detection position in the target sequencecan be determined by denaturing the ligation complex and adding theligated strand to an electrode comprising a capture probe and aself-assembled monolayer and detecting electron transfer between the ETMpresent on the ligated strand and the electrode.

Alternatively, a target nucleic acid comprising a first domain capableof binding in a sequence-specific manner to a first ligation probe, asecond domain capable of binding in a sequence-specific manner to asecond ligation probe, and a detection position is contacted with afirst ligation probe, and at least a second ligation probe comprising aninterrogation position. In this embodiment, the identity of the baselocated at the interrogation position can be determined by denaturingthe ligation complex and adding the ligated strand to an electrodecomprising a capture probe, a self-assembled monolayer, and a labelprobe comprising a first domain that is capable of binding in asequence-specific manner the ligated strand and a second domaincomprising at least one ETM. Electron transfer between the ETMs presenton the label probe and the electrode is an indication of the presence ofthe ligated strand comprising the base or sequence of interest.

In other embodiments, one ligation probe can be attached to an electrodeusing a capture probe. The attached ligation probe is contacted with oneor more additional ligation probes comprising one or more ETMs and atarget nucleic acid comprising a detection position. If the base locatedat the interrogation position on one of the ligation probes is capableof pairing in a sequence-specific manner with the base located at thedetection position on the target nucleic acid, a ligation complexcomprising the target nucleic acid and a ligated strand comprising theattached ligation probe and a second ligation probe is formed. Theligation complex is denatured and the base located at the detectionposition is identified by detecting electron transfer between the ETMpresent on the ligated strand and the electrode.

Other configurations also are possible for detecting a base or sequenceof interest. For example, rather than attaching the ETMs to the ligationprobes, the ETMs may be attached to a label probe. Followingdenaturation of the ligation complex, the ligated strand can becontacted with the label probe and detection of electron transferbetween the ETMs on the label probe and the electrode used as anindication of the presence of the base of interest.

The compositions and methods of the present invention can be used in avariety of contexts. In a preferred embodiment, the compositions andmethods of the present invention may be used to detect SNPs in a singlenucleic acid or in a plurality of nucleic acids. In the latterembodiment, arrays of electrodes, each comprising a ligation probe witha different interrogation position are used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the use of ligation probes to identify anucleotide at a detection position within a target sequence. FIG. 1Aillustrates the formation of a ligated strand when there are nomismatched nucleotides. In FIG. 1A, a target sequence 5 hybridized to afirst 20 and a second 21 ligation probe. As outlined herein, either thefirst 20 or second 21 ligation probe comprises an electron transfermoiety 3 and a nucleotide at the interrogation 15 position that willhybridize to a nucleotide at the detection 10 position of the targetsequence 5. If there are no mismatched nucleotide(s) present, a ligatedstrand 6 comprising the first and second ligation probe will be formed.FIG. 1B illustrates the situation when a mismatched nucleotide(s) 16 ispresent at the interrogation position. In this case, a ligated strand isnot formed.

FIGS. 2A and 2B depict two embodiments of the present invention. FIG. 2Aillustrates the use of a first ligation probe 20 comprising a 5′-iodidemoiety, and a second ligation probe 21 comprising a 3′-sulfur moiety andan ETM 3. FIG. 2B illustrates the use of a first ligation probe 20comprising a 5′-iodide moiety and a second ligation probe 21 comprisinga 3′-sulfur moiety and a label sequence 2.

FIGS. 3A through 3E depict the formation of a ligation complexcomprising a target sequence 5 and one, two, three and/or four ligationprobes 21, 22, 23 and 24, each containing a different label 3, 7, 8, and9 and a different nucleotide N_(A), N_(B), N_(C), and N_(D) at theinterrogation 15 position.

FIG. 4 depicts non enzymatic chemical ligation using a 3′-phosphothioateand 5′-iodo group.

FIGS. 5A and 5B depict activation of the terminal 5′-phosphate withN-cyanoimidazole or EDC.

FIG. 6 depicts attachment of multiple ETMs to a portion of a ligationprobe. FIG. 6 illustrates a ligation probe 20 comprising a first portion27 that hybridizes to a portion of the target sequence 5 and a secondportion 28 comprising ETMs 3.

FIG. 7 depicts the use of a capture sequence to identify the base at thedetection position. Target sequence 5 with detection position 10 isadded to first ligation probe 20 with interrogation position 15 andcapture sequence 30 and a second ligation probe 21 with a label 35(although a label sequence could also be used, or, in the case ofreporterless sensing, no label or label probe). If the interrogationbase 15 is complementary to the detection position 10, ligation willoccur, the ligated complex is denatured, and the ligated sequence isadded to an array comprising an electrode 1 with a SAM 50 and captureprobe 40 attached via an attachment linker 45. As described herein,other detection arrays can also be used.

FIGS. 8A-8C depict three preferred embodiments of the invention. In FIG.8A, target sequence 5 with detection position 10 is added to firstligation probe 20 with interrogation position 15 and a second ligationprobe 21. If the interrogation base 15 is complementary to the detectionposition 10, ligation will occur, the ligated complex is denatured,unreacted probes can be optionally removed and the ligated sequence isadded to an array comprising an electrode 1 with a SAM 50 and captureprobe 40 attached via an attachment linker 45. A label probe 55 withlabels 35 (although again, these may not be required in someembodiments) is then added, which has a first portion complementary to adomain of the ligated probe. As described herein, other detection arrayscan also be used. FIGS. 8B and 8C depict situations wherein one of theligation probes serves as the capture probe. FIG. 8B shows the situationwhere one of the ligation probes has a label. FIG. 8C utilizes a labelprobe 55, with a first portion 60 that hybridizes to the ligated probeand a recruitment linker 75 with labels 35.

FIG. 9 depicts electron transfer moieties with different redoxpotentials that may be used as labels in the methods of the presentinvention.

FIG. 10 depicts the starting materials and autoligation using EDCdescribed in Example 1.

FIGS. 11A-D depict ligation efficiency using various incubation times.

FIG. 12 depicts electrochemical detection of SNPs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods ofdetermining the sequence of a target nucleic acid, using electrochemicaldetection on an electrode. The invention preferably includes theidentification of a single nucleotide in a target nucleic acid, i.e.,the detection of single nucleotide polymorphisms (SNPs).

The invention utilizes a non enzymatic, reagent free method for theligation of oligonucleotides. In this method, two oligonucleotidestrands bound at adjacent sites on a complementary target strand,undergo autoligation by displacement of a 5′-end iodide moiety with a3′-end sulfur moiety. This reaction occurs with substantial specificityas to allow the detection of single base mismatches at either side ofthe ligation junction, as well as a few nucleotides away from theligation junction. Ligation does not occur if mismatches are present.

In general, ligation probes are incorporated into a ligation complex,comprising a target molecule and a label, such as an electron transfermoiety (ETM). Additionally, the ligation probes are labeled at the3′-end with a sulfur containing moiety or at the 5′-end with an iodidemoiety. If there are no base mismatches between the target and theligation probes, the ligation reaction proceeds efficiently, yielding aligated strand. The ligation complex is denatured and added to anelectrode comprising a capture probe. The presence or absence of theligated strand is detected by monitoring the passage of electronsbetween the ETM and the electrode.

The presence or absence of the ETMs are detected as described below andin U.S. Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348 and5,780,234; U.S. Ser. Nos. 08/911,589; 09/135,183; 09/306,653;09/134,058; 09/295,691; 09/238,351; 09/245,105; 09/338,726; and09/626,096; and PCT applications WO98/20162; WO 00/16089; PCTUS99/01705; PCT US99/01703; PCT US00/10903 and PCT US99/10104, all ofwhich are expressly incorporated herein by reference.

Accordingly, the present invention provides compositions and methods fordetecting the presence or absence of a target sequence. By “targetsequence” herein is meant a nucleobase sequence on a nucleic acid soughtto be detected. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, ora product of an amplification process using a nucleic acid provided bynature or by a synthetic process. As is outlined herein, the targetsequence may be a target sequence from a sample, or a secondary targetsuch as the product of a chemical ligation reaction outlined herein. Itmay be any length, with the understanding that longer sequences are morespecific. As will be appreciated by those in the art, the complementarytarget sequence may take many forms. For example, it may be containedwithin a larger nucleic acid sequence, i.e. all or part of a gene ormRNA, a restriction fragment of a plasmid or genomic DNA, among others.

Each target sequence comprises a region of unique nucleobase sequencethat may be used to discriminate one target sequence from another targetsequence. The nucleobase sequence may comprise a single base, two ormore contiguous bases, or two or more non contiguous bases. “Nucleobase”means those naturally occurring and those synthetic nitrogenous,aromatic moieties commonly found in the nucleic acid arts. Examples ofnucleobases include purines and pyrimidines, genetically encodednucleobases, analogs of genetically encoded nucleobases, and purelysynthetic nucleobases. Specific examples of genetically encoded basesinclude adenine, cytosine, guanine, thymine, and uracil. Specificexamples of analogs of genetically encoded bases and synthetic basesinclude 5-methylcytosine, pseudoisocytosine, 2-thiouracil and2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine),N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine),N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).5-propynyl-uracil, 2-thio-5-propynyl-uracil. Other non-limiting examplesof suitable nucleobases include those nucleobases illustrated in FIGS.2(A) and 2(B) of U.S. Pat. No. 6,357,163, incorporated herein byreference in its entirety.

Nucleobases can be linked to other moieties to form nucleosides,nucleotides, and nucleoside/tide analogs. As used herein, “nucleoside”refers to a nucleobase linked to a pentose sugar. Pentose sugars includeribose, 2′-deoxyribose, 3′-deoxyribose, and 2′,3′-dideoxyribose.“Nucleotide” refers to compound comprising a nucleobase, a pentose sugarand a phosphate. Thus, as used herein a nucleotide refers to a phosphateester of a nucleoside, e.g., a triphosphate. Nucleic acid analogs,including nucleoside and nucleotide analogs, are described below.

The target sequence further comprises different target domains; forexample, a first target domain of the target sequence may hybridize to afirst ligation probe and a second target domain may hybridize to asecond ligation probe. As more fully outlined below, the target sequencemay also have a domain that hybridizes to a capture probe. The targetdomains may be adjacent or separated by one or more nucleotides. Unlessspecified, the terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

The target sequence comprises a position for which sequence informationis desired, generally referred to herein as the “detection position”. Ina preferred embodiment, the detection position is a single nucleotide,although in some embodiments, it may comprise a plurality ofnucleotides, either contiguous with each other or separated by one ormore nucleotides. By “plurality” is meant two or more nucleotides.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinis meant at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with bicyclic structuresincluding locked nucleic acids (LNAs), Koshkin et al., J. Am. Chem. Soc.120:13252-3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997,page 35. All of these references are hereby expressly incorporated byreference. The modifications of the ribose-phosphate backbone may bedone to facilitate the addition of ETMs, or to increase the stabilityand half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred nucleic acid analogs are peptide nucleic acids(PNA), and peptide nucleic acid analogs. “Peptide Nucleic Acid” or “PNA”refers to nucleic acid analogs in which the nucleobases are attached toa polyamide backbone through a suitable linker (i.e. methylene carbonyl,aza nitrogen) such as described in any one or more of U.S. Pat. Nos.5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,5,986,053, 6,107,470, 6,451,968, 6,441,130, 6,414,112 and 6,403,763; allof which are incorporated herein by reference. PNA backbones aresubstantially non-ionic under neutral conditions, in contrast to thehighly charged phosphodiester backbone of naturally occurring nucleicacids. This results in two advantages. First, the PNA backbone exhibitsimproved hybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration. This is particularlyadvantageous in the systems of the present invention, as a reduced salthybridization solution has a lower Faradaic current than a physiologicalsalt solution (in the range of 150 mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. Other preferred embodiments utilize diaminopurines(see e.g., Haaima et al., 1997, Nucleic Acids Res., 25: 4639-4643; andLohse et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 11804-11808).

The nucleic acid comprising the target sequence may be provided from anysource. For example, the nucleic acid to be analyzed may be isolated orenriched from a sample, or be present in a cell or a tissue. As will beappreciated by those skilled in the art, in addition to the nucleicacid, the sample may comprise any number of other things, including, butnot limited to, bodily fluids (including, but not limited to, blood,urine, serum, lymph, saliva, anal and vaginal secretions, perspirationand semen, of virtually any organism, with mammalian samples beingpreferred and human samples being particularly preferred); environmentalsamples (including, but not limited to, air, agricultural, water andsoil samples); biological warfare agent samples; research samples (i.e.in the case of nucleic acids, the sample may be the products of anamplification reaction, including both target and signal amplificationas is generally described in PCT/US99/01705, such as PCR amplificationreaction); purified samples, such as purified genomic DNA, RNA,proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc). As willbe appreciated by those in the art, virtually any experimentalmanipulation may have been done on the sample.

If required, the target sequence is prepared using known techniques. Aswill be appreciated by those skilled in the art, the sample may betreated to lyse the cells, using known lysis buffers, electroporation,etc., with purification and/or amplification as needed. Examples ofamplification processes that can be the source for the target sequenceinclude, but are not limited to, Polymerase Chain Reaction (PCR), LigaseChain Reaction (LCR), Strand Displacement Amplification (SDA; see, e.g.,Walker et al., 1989, PNAS 89:392-396; Walker et al., 1992, Nucl. AcidsRes. 20(7):1691-1696; Nadeau et al., 1999, Anal. Biochem.276(2):177-187; and U.S. Pat. Nos. 5,270,184, 5,422,252, 5,455,166 and5,470,723), Transcription-Mediated Amplification (TMA), Q-beta replicaseamplification (Q-beta), Rolling Circle Amplification (RCA), Lizardi,1998, Nat. Genetics 19(3):225-232 and U.S. Pat. No. 5,854,033),Asymmetric PCR (Gyllensten and Erlich, 1988, PNAS, 85:7652-7656) orAsynchronous PCR (see, e.g., WO 01/94638).

In a preferred embodiment, a step in the methods of the inventioninclude a step to produce an excess of one strand over the other. Aswill be appreciated by those in the art, a variety of methods can beused, including, but not limited to, asymmetric polymerase chainreaction (APCR), exonuclease methods and the capture of the non-targetstrand.

In a preferred embodiment, asymmetric polymerase chain reaction (APCR)is used to enhance the production of the single stranded nucleic acidfragment used as the target sequence for detection as outlined herein.Traditional APCR techniques produces a single stranded bias by using theprimers in a ratio of 100 to 1, although a variety of ratios rangingfrom 10:1 to 100:1 can be used as well (see, e.g., Gyllensten andErlich, 1988, PNAS, 85:7652-7656; WO 00/20476; the disclosures of whichare incorporated herein by reference in their entirety).

In a preferred embodiment, a novel nested primer method is used toamplify the patient sample. In this embodiment, an enhancement of targetproduction is achieved using a two step process: a first symmetric PCRstep, using a 1:1 ratio of primers, followed by the addition (preferablyto the same reaction) of a second APCR step, using a ratio of 50:1(again, with ratios of from about 10:1 to over 100:1 being useful).Alternatively, these reactions may be done in two steps as well. Thishas been shown to result in a 3-6 fold increase over a one step APCRreaction.

In a preferred embodiment, the asymmetric amplification step isaccomplished as described in WO 00/20476, the disclosure of which isincorporated herein by reference in its entirety.

Accordingly, the compositions and methods of the present invention areused to identify the nucleotide(s) at the detection position located ona target sequence using ligation probes. The ligation probes aredesigned to “bind” or “hybridize” to target sequences that arecomplementary, such that double-stranded hybrids are formed between theligation probes and the target sequence. “Complementary refers tosequences that contain no mismatched base pairs, e.g., A and T, A and U,C and G. “Binding” or “hybridization” refers to the base-pairinginteractions of one nucleic acid strand with another that results in theformation of a double-stranded structure, a triplex structure or aquaternary structure.

As will be appreciated by a person skilled in the art, the probes alsocan be designed to bind to sequences that are “substantiallycomplementary”, “identical”, or “substantially identical”. Two singlestranded nucleic acid molecules are said to be substantiallycomplementary when the nucleobases of one strand, optimally aligned andcompared with the nucleobases of the other strand differ by onemismatched base pair, i.e., A and C, A and G, T and C, T and G. Twosingle stranded nucleic acid molecules are said to be “identical” whenthe nucleobases of one strand, optimally aligned and compared with thenucleobases of the other strand are the same, i.e., A and A, C and C, Gand G. “Substantially identical” refers to sequences that contain 1 basethat is not identical, i.e., A and C.

Nucleic acid samples that do not exist in a single-stranded state in theregion of the target sequence(s) are generally rendered single-strandedin such region(s) prior to detection or hybridization. Generally,nucleic acid samples will be rendered single-stranded in the region ofthe target sequence using heat denaturation. For polynucleotidesobtained via amplification, methods suitable for generatingsingle-stranded amplification products are preferred. Non-limitingexamples of amplification processes suitable for generatingsingle-stranded amplification product polynucleotides include, but arenot limited to, T7 RNA polymerase run-off transcription, RCA, AsymmetricPCR (Bachmann et al., 1990, Nucleic Acid Res., 18,1309), andAsynchronous PCR (WO 01/94638). Commonly known methods for renderingregions of double-stranded polynucleotides single stranded, such as theuse of PNA openers (U.S. Pat. No. 6,265,166), may also be used togenerate single-stranded target sequences on a polynucleotide.

The probes can be virtually any nucleic acid or nucleic acid analog thatis capable of binding to a target sequence in a sequence-specificmanner. Thus, probes useful in the invention include, but are notlimited to DNA, RNA, PNA, and LNA, or mixtures, such as, PNA linked toLNA, LNA linked to DNA, or LNA linked to RNA. In preferred embodiments,the probes are DNA.

By “ligation probe” herein is meant a single stranded nucleic acidmolecule that has a sequence that is complementary to a target specificsequence. A minimum of two ligation probes, a first and second ligationprobe, are required to detect a single nucleotide polymorphism (SNP) ora sequence comprising two or more bases in a target sequence. In otherembodiments, up to five ligation probes can be used, i.e. a first,second, third, fourth, and fifth ligation probes (see FIG. 3E). Unlessspecified, the terms “first”, “second”, “third”, “fourth” and “fifth”are not meant to confer an orientation as to which probe binds to whichdomain of the target sequence or which probe(s) have an interrogationposition. In genotyping embodiments, at least one of the ligation probescomprises an interrogation position. By “interrogation position” hereinis meant the base that base pairs with the detection position base inthe target sequence.

The ligation probes comprise reactive groups to facilitate chemicalligation. Reactive groups include sulfur moieties, and leaving groups,such as iodide and tosylate (see, e.g., Xu and Kool, 1999, Nucleic AcidsResearch, 27(3): 875-881; the disclosure of which is incorporated hereinby reference in its entirety). In a preferred embodiment, the leavinggroup is a 5′-iodide moiety. Preferably, modified nucleotides comprisinga 5′ iodide moiety are incorporated during the solid phase synthesis ofthe ligation probe. See, for example, Xu and Kool, describing the solidphase synthesis of an oligonucleotide using a commercially available5-iodothymidine phosphoramidite reagent (Glen Research; Xu, Y. and Kool,E. T., (1997) Tetrahedron Letters, 38:5595). A generalized structure forligation probes comprising a 5′-iodide moiety is shown in structure 1:

In a preferred embodiment one of the ligation probes comprise a3′-sulfur moiety. The sulfur group can be incorporated into anoligonucleotide strand using a phosphorothioate group (see Herrlein, M.K., et al., (1997) J. Am. Chem. Soc., 117:10151; Xu, Y. and Kool, E. T.,(1997) Tetrahedron Letters, 38:5595; the disclosures of which areincorporated herein by reference). Sulfur groups also can beincorporated into an oligonucleotide strand using dithiophosphate. Ageneralized structure for ligation probes comprising a 3′-sulfur moietyis shown in structure 2:

Unless specified, the terms “first”, “second”, “third”, “fourth” and“fifth” are not meant to confer an orientation of the sulfur and iodidemoieties. For example, the first ligation probe may be labeled at its 5′end with an iodide moiety or at its 3′end with a sulfur moiety.Similarly, the second ligation probe may be labeled at its 5′-end withan iodide moiety or at its 3′-end with a sulfur moiety. The same is truefor any of the probes used in the methods of the invention.

In some embodiments, the 5′- and 3′ moieties are hydroxyls. In thisembodiment, a ligated strand comprising a first and second ligationprobe may be formed by activating the terminal 5′-phosphate in thepresence of a coupling reagent. Suitable coupling reagents includeN-cyanoimidazole (Kanaya, R. and Yanagawa, H., (1986) Biochemistry,25:7423; Luebke, K. J. and Dervan, P. B., (1989) J. Am. Chem. Soc.,111:8733), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC) (Metelev, V. G., et al., (1999) Nucleosides & Nucleotides18:2711), BrCN (Shabarova et al., 1991, Nucleic Acids Research,19(15):4247-4251), and K₃Fe(CN)₆ or Kl₃ (Gryaznoz, S. M. and Letsinger,R. L., (1993) Nucleic Acids Research, 21:1403; the disclosures of whichare incorporated herein by reference).

In a preferred embodiment, a terminal 5′-phosphate is activated in thepresence of a coupling reagent. FIGS. 5A and 5B depict activation of theterminal 5′-phosphate with N-cyanoimidazole or EDC.

FIG. 4 depicts non enzymatic chemical ligation using a 3′-phosphothioateand 5′-iodo group. As shown in FIG. 4, the 3′-sulfur moiety displacesthe 5′-iodide moiety resulting in a joined, i.e. ligated, nucleic acidstrand. This reaction occurs without the need for added reagents. Theresulting ligated strand differs from natural DNA by the replacement ofa single oxygen atom with a single sulfur atom. As a result of thereplacement of the single oxygen atom with a single sulfur atom, abridging 5′-phosphorothioester is formed. The bridging5′-phophorothioester is stable in solution, resistant to enzymatichydrolysis and does not affect the ability of polymerases to replicateor transcribe the ligated strand (see, e.g., Xu and Kool, 1999, NucleicAcids Research, 27(3):875-881).

One or more of the ligation probes can also include a detectable label.By “label” or “detectable label” herein refers to a moiety that, whenattached to a probe of the invention, renders such a probe detectableusing known detection methods, e.g., electronic, spectroscopic,photochemical, or electrochemiluminescent methods. Exemplary labelsinclude, but are not limited to, electron transfer moieties (ETMs).

In a preferred embodiment, at least one of the ligation probes comprisesa first ETM. By “ETM”, “electron donor moiety”, “electron acceptormoiety”, or grammatical equivalents herein refers to molecules capableof electron transfer under certain conditions. It is to be understoodthat electron donor and acceptor capabilities are relative; that is, amolecule which can lose an electron under certain experimentalconditions will be able to accept an electron under differentexperimental conditions. It is to be understood that the number ofpossible electron donor moieties and electron acceptor moieties is verylarge, and that one skilled in the art of electron transfer compoundswill be able to utilize a number of compounds in the present invention.Preferred ETMs include, but are not limited to, transition metalcomplexes, organic ETMs, and electrodes. See, e.g., U.S. Pat. Nos.6,096,273, 6,264,825, and 6,221,583 for a discussion of suitable ETMs,the disclosures of which are incorporated herein by reference.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventionare described, for example, in U.S. Pat. Nos. 6,096,273, 6,264,825, and6,221,583 the disclosures of which are incorporated herein by reference.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules are described, for example, inU.S. Pat. Nos. 6,096,273, 6,264,825, and 6,221,583, the disclosures ofwhich are incorporated herein by reference.

Preferred ETMs are metallocenes, with ferrocene and ferrocenederivatives being particularly preferred. See, e.g., U.S. Pat. Nos.6,096,273, 6,264,825, and 6,221,583, and U.S. Publication No.20030143556, the disclosures of which are incorporated herein byreference.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, in some embodiments, redox proteinsare not preferred.

In one embodiment, ETMs, such as Ru²⁺(bpy)₃, can be used for detectionmethods based on electrochemiluminscence (see Blackburn, 1991, ClinChem, 37:1534-1539). However, in some embodiments, ETMs are notpreferred for use in detection methods based on electrochemiluminscence.

The choice of the specific ETMs will be influenced by the nature of theassay, i.e., detection of SNPs with two or more alleles, and by the typeof electron transfer detection used. For the detection of SNPs with twoor more alleles, ETMs with different redox potentials are used (see U.S.Publication No. 20030143556, the disclosure of which is incorporatedherein by reference). The redox potentials of the ETMs are chosen suchthat they are distinguishable in the assay system used. By “redoxpotential” (sometimes referred to as E₀) herein is meant the voltagewhich must be applied to an electrode (relative to a standard referenceelectrode such as a normal hydrogen electrode) such that the ratio ofoxidized and reduced ETMs is one in the solution near the electrode. Ina preferred embodiment, the redox potentials are separated by at least100 mV, although differences either less than this or greater than thismay also be used, depending on the sensitivity of the system, theelectrochemical measuring technique used and the number of differentlabels used.

In a particularly preferred embodiment, derivatives of ferrocene areused; for example, ferrocenes with or without ring substituents, i.e.,the addition of an amine or an amide, a carboxylate, a halogen, etc.FIG. 9 illustrates ferrocene derivatives with different redoxpotentials. See also U.S. Publication No. 20030143556 for a discussionof ETMs with distinguishable redox potentials, the disclosure of whichis incorporated herein by reference in its entirety.

In a preferred embodiment, each ligation probe having an interrogationposition with a different base, has a covalently attached ETM with adistinguishable redox potential.

In a preferred embodiment, a plurality of ETMs are used on a singleligation probe. The use of multiple ETMs provides signal amplification,and thus, allows more sensitive detection limits. While the use ofmultiple ETMs on nucleic acids that hybridize to complementary strandscan decrease the Tms of the hybridization complexes depending on thenumber, site of attachment and spacing between the multiple ETMs, thisis not a factor when the ETMs are localized to a portion of the probewhich does not hybridize to a complementary sequence (see FIG. 6). Theportion of the probe that includes the ETMs, but lacks a complementarysequence for a target nucleic acid or another nucleic acid molecule usedin the methods of the present invention is referred to herein as a“recruitment linker”.

In a preferred embodiment, multiple ETMs are used on a recruitmentlinker, since this linker does not hybridize to a complementarysequence. Preferably, the recruitment linker is nucleic acid, or nucleicacid analog. Pluralities of ETMs are preferred, with at least about 2ETMs per recruitment linker being preferred, and about 8 beingparticularly preferred, and at least about 10 to 50 being preferredinsome cases. In some instances, very large numbers of ETMs, i.e. 50 to1000, can be used. In general, the number chosen depends on the system,the required sensitivity, and the solubility of the ETMS, etc. In thecase of ferrocenes generally from 4-10 per probe are used, with 8 beingparticularly preferred.

The probes of the present invention may be synthesized using routinemethods. For example, methods of synthesizing oligonucleotide probes aredescribed in U.S. Pat. No. 4,973,679; Beaucage, 1992, Tetrahedron48:2223-2311; U.S. Pat. No. 4,415,732; U.S. Pat. No. 4,458,066; U.S.Pat. No. 5,047,524 and U.S. Pat. No. 5,262,530; the disclosures of whichare incorporated herein by reference. The synthesis may be accomplishedusing automated synthesizers available commercially, for example theModel 392, 394, 3948 and/or 3900 DNA/RNA synthesizers available fromApplied Biosystems, Foster City, Calif.

Methods of synthesizing labeled probes with ETMS attached via: (1) abase; (2) the backbone, including the ribose, the phosphate, orcomparable structures in nucleic acid analogs; (3) nucleosidereplacement, or (4) metallocene polymers, are also well-known. Asspecific examples, see WO 00/20476 and U.S. Publication Nos. 20020121314and 20030143556; the disclosures of which are incorporated herein byreference.

The basic principle of the chemical ligation assay for the detection ofSNPs is illustrated in FIGS. 3A-3E. FIGS. 3A-C depict an embodiment fordetection of a SNP that exists as two different alleles. In thisembodiment, three probes are used. One of the three probes, i.e. 20,lacks an interrogation base and ETM, but includes a sequence that iscomplementary to one of the two domains of the target strand 5. Theother two probes, 21 and 22, include an interrogation position 15 andare labeled with ETMs having two different redox potentials, redoxpotential 3 and redox potential 7. As shown in FIG. 3B, a ligated strandcomprising ligation probes 20 and 21 and ETM 3 is formed if the baseN_(A) at interrogation position 15 on probe 21 is complementary to thebase N_(A) at the detection position 10 on the target sequence. If thebase N_(A) at interrogation position 15 on probe 21 is not complementaryto the base N_(B) at the detection position 10 on the target strand, aligated strand comprising ligation probes 20 and 21 is not formed.However, as shown in FIG. 3C, a ligated strand comprising ligationprobes 20 and 22 and ETM 7 is formed if the base N_(B) at interrogationposition 15 on probe 22 is present. Detection of ETM 3 or 7 is used toidentify which base is present at the detection position on the targetstrand 5.

FIG. 3D illustrates an embodiment for detecting a SNP with 3 alleles. Inthis embodiment, 4 ligation probes are used. A first probe 20, having asequence complementary to the target strand 5, and three ligationprobes, 21, 22, and 23, having a sequence complementary to a differentdomain of the target strand 5, an interrogation position 15, and ETMswith different redox potentials, 3, 7 and 8. A ligated strand,comprising ligation probe 20 and one of the other three probes 21, 22and 23, will be formed depending on which probe has a complementary baseat interrogation position 15 to the base present at detection position10. For example, if the base N_(C) is present at the detection position,than a ligated strand comprising ligation probe 20 and 23 will beformed. Similarly if base N_(B) is present at the detection position,than a ligated strand comprising ligation probes 20 and 22 will beformed. Likewise, if base N_(A) is present at the detection position,than a ligated strand comprising ligation probes 20 and 21 will beformed.

FIG. 3E illustrates an embodiment for detecting a SNP with 4 alleles. Inthis embodiment, 5 ligation probes are used. A first probe 20, having asequence complementary to the target strand 5, and three ligationprobes, 21, 22, 23, and 24, having a sequence complementary to adifferent domain of the target strand 5, an interrogation position 15,and ETMs with different redox potentials, 3, 7 8, and 9. A ligatedstrand, comprising ligation probe 20 and one of the other four probes21, 22, 23, and 24, will be formed depending on which probe has acomplementary base at interrogation position 15 to the base present atdetection position 10. For example, if the base N_(D) is present at thedetection position, than a ligated strand comprising ligation probe 20and 24 will be formed. If, on the other hand, the base N_(C) is presentat the detection position, than a ligated strand comprising ligationprobe 20 and 23 will be formed. Similarly if base N_(B) is present atthe detection position, than a ligated strand comprising ligation probes20 and 22 will be formed. Likewise, if base N_(A) is present at thedetection position, than a ligated strand comprising ligation probes 20and 21 will be formed. Thus, by using different probes, each with adifferent base at the interrogation position and each with a differentlabel, the identification of the base at the detection position can beelucidated.

Accordingly, the ligation substrates of the invention can take on anumber of configurations.

By “ligation substrate” herein is meant a substrate for chemicalligation comprising at least one target nucleic acid strand and two ormore ligation probes. For example, as shown in FIG. 1A, the ligationsubstrate comprises target strand 5 and ligation probes 20 and 21. Oncea ligation substrate is formed, and chemical ligation occurs, a“ligation complex” is formed, i.e., the structure depicted in FIG. 1A,comprising target strand 5 and ligated strand 6. As used herein,“ligated strand” refers to the nucleic acid strand formed by joining twoligation probes together using the methods of the present invention.

In a preferred embodiment, the ligation substrate comprises at least onetarget nucleic acid strand and two ligation probes, one of whichcomprises an interrogation position and either a label or a labelsequence to which a label probe binds, as described below (see FIGS.3A-3C). Alternatively, at least one target nucleic acid strand and threeor more ligation probes are used (see FIGS. 3D and 3E). In thisembodiment, the first ligation probe is complementary to the firstdomain of the target sequence, and the other ligation probes comprisedifferent bases at the interrogation position, as well as differentlabels or different label sequences. Only when the correct ligationprobes (e.g. with the perfectly complementary sequence) hybridize to thetarget nucleic acid strand will ligation occur.

As will be appreciated by a person skilled in the art, more than onedetection position can be evaluated using ligation probes with differentbases at the interrogation position and distinguishable labels. Forexample, in FIG. 3E, target nucleic acid strands with different bases atthe detection position, i.e. a target strand with base N_(A), a targetstrand with base N_(B), a target strand with base N_(C) and a targetstrand with base N_(D) can be added to a ligation complex comprisingseveral copies of ligation probes 20, 21, 22, 23 and 24. Formation ofligated strands can be detected based on the redox potentials of theETMs, i.e., a ligated strand comprising ligation probes 20 and 21 andETM 3, a ligated strand comprising ligation probes 20 and 22, and ETM 7,etc.

Accordingly, in a preferred embodiment, the ligation substrate comprisesa target nucleic acid strand and two ligation probes. As is depicted inFIG. 2A, the first ligation probe can comprise an iodide moiety and thesecond ligation probe can comprise the interrogation position, thesulfur moiety, and a label (FIG. 2A) or a label sequence (FIG. 2B) towhich a label probe will hybridize. Either probe may also comprise anoptional capture sequence for subsequent hybridization to acomplementary sequence present on a capture probe, as outlined below.

As is shown in FIG. 1A, the ligation substrate comprises a targetnucleic acid strand with a first domain that hybridizes to a firstligation probe and a second domain that hybridizes to a second ligationprobe. If the second ligation probe comprises an interrogation positionwith a base complementary to the base located at the detection position,i.e. (N_(C)), on the target strand, and the first ligation probe issubstantially complementary to the first domain, a ligation complex isformed comprising the target sequence and a ligated strand consisting ofthe two ligation probes. If, however, as shown in FIG. 1B, the secondligation probe comprises an interrogation position with a base that isnot complementary to the base located at the detection position, i.e.Nx, a ligated strand will not be formed.

In a preferred embodiment, the invention provides different ligationprobes with different bases and different redox labels. This isanalogous to the “two color” or “four color” idea of competitivehybridization and is also analogous to sequencing by hybridization. Forexample, sequencing by hybridization has been described (Drmanac et al.,Genomics 4:114 (1989); Koster et al., Nature Biotechnology 14:1123(1996); U.S. Pat. Nos. 5,525,464; 5,202,231 and 5,695,940, among others,all of which are hereby expressly incorporated by reference in theirentirety).

For example, the methods of the present invention can use a ligationsubstrate comprising a target strand and three ligation probes. Thefirst ligation probe 20 is substantially complementary to either thefirst or second domain of the target sequence. The second ligation probe21 comprises an interrogation position with a first base and a firstETM. The third ligation probe 22 comprises an interrogation positionwith a second base and a second ETM. A ligation complex will be formedif either the second or third probe comprises an interrogation positionwith a base that is complementary to the base at the detection positionon the target strand. Thus, a ligation complex comprising the targetstrand and the first and second ligation probes may be formed (FIG. 3B).Alternatively, a ligation complex comprising the target strand and thefirst and third ligation probes may be formed (FIG. 3C).

In other preferred embodiments, a ligation substrate comprising a targetstrand, a first ligation probe 20 substantially complementary to eitherthe first or second domain of the target strand, second 21, third 22 andfourth 23 ligation probes comprising interrogation positions and ETMs isused (FIG. 3D). The second ligation probe comprises an interrogationposition with a first base and a first ETM, the third ligation probecomprises an interrogation position with a second base and a second ETM,and the fourth ligation probe comprises an interrogation position with athird base and a third ETM. Thus, a ligation complex comprising thefirst ligation probe and either the second, third or fourth ligationprobes is possible depending on which probe comprises an interrogationposition with a base that is complementary to the base at the detectionposition on the target strand (FIG. 3D).

In a preferred embodiment, a ligation substrate comprising a targetstrand, a first ligation probe 20 substantially complementary to eitherthe first or second domain of the target strand, second 21, third 22,fourth 23, and fifth 24 ligation probes comprise interrogation positionsand ETMs is used (FIG. 3E). The second ligation probe comprises aninterrogation position with a first base and a first ETM, the thirdligation probe comprises an interrogation position with a second baseand a second ETM, the fourth ligation probe comprises an interrogationposition with a third base and a third ETM, and the fifth ligation probecomprises an interrogation position with a fourth base and a fourth ETM.Thus, a ligation complex comprising the first ligation probe and eitherthe second, third, fourth, or fifth ligation probe is possible dependingon which probe comprises an interrogation position with a base that iscomplementary to the base at the detection position on the targetstrand.

Accordingly, the ligation probes are hybridized to the target sequenceto form a ligation complex. This method is based on the fact that twooligonucleotides hybridized at adjacent sites on a target strand undergochemical ligation by displacement of a 5′-end iodide moiety with a3″-end sulfur moiety, if complementarity exists at the two bases beingligated together. Thus, in this embodiment, the target sequencecomprises a contiguous first target domain adjacent to a second targetdomain comprising the detection position. That is, the detectionposition is “between” the first target domain and the rest of the secondtarget domain. A first ligation probe is hybridized to the first targetdomain and a second ligation probe is hybridized to the second targetdomain. If the second ligation probe has a base complementary to thedetection position base, and the adjacent base on the first probe iscomplementary to the corresponding base on the target strand, chemicalligation will proceed resulting in the ligation of the first and secondligation probes such that a ligated strand or probe is formed. If thiscomplementarity does not exist, a ligated strand is not formed.

The ligation substrate can be unbound, or bound to a solid support. Forexample, the target strand, and all of the ligation probes can be addedto a suitable ligation buffer and incubated for a period of timesufficient for the formation of a ligation complex (see FIG. 8A).Alternatively, a component of the ligation substrate may be attached toa solid support. For example, as illustrated in FIG. 8B or 8C, one ofthe ligation probes can be attached to an electrode via an attachmentlinker, and the other ligation probe and target strand free in solution.

The efficiency of ligation will vary depending on whether apolymorphism, i.e., mismatch occurs at the detection position. Aligation complex with no mismatch at the detection position (i.e.“matched complex”) can be distinguished from a mismatched ligationcomplex by increasing the temperature, as a “matched” ligation complexis more stable than a complex comprising a mismatch. Depending on thelocation of the mismatch, (e.g. proximity to the ligation junction) andthe type of probe (e.g. padlock probe) temperatures between 0° C. to 70°C. are used to distinguish mismatched ligation complexes from matchedligation complexes. See Xu, Y. and Kool, E. T., (1999) Nucleic AcidsResearch, 27:875; Metelev, V. G., et al., (1999) Nucleosides &Nucleotides, 18:2711; and FIGS. 11A-D, and 12.

Conditions for efficient ligation are known to those of skill in the art(see, e.g., Xu and Kool, 1997, Tetrahedron Letters, 38(32):5595-5598.Variables that may be varied to optimize ligation conditions includelocation of the mismatch temperature, incubation time, target/probeconcentrations, salt concentration, pH, as well as other components ofthe ligation buffer. For example, temperatures between 16° C. and 23° C.may be used, and ligation reactions may be incubated between 2 to 20hours.

In some embodiments, heat cycling is used to allow the ligated strand tobe denatured off the target sequence such that it may serve as atemplate for further reactions. In these embodiments, temperaturesbetween 92° C. to 95° C. are used to denature the ligated strand/targetstrand complex.

In a preferred embodiment, the ligated strand is disassociated from thetarget sequence using heat denaturation and added to an assay complex.By “assay complex” herein is meant the collection of hybridizationcomplexes comprising nucleic acids that contain at least one ETM andthus allow detection. The composition of the assay complex depends onthe use of the different probe components outlined herein. For example,as illustrated in FIG. 8A, the assay complex can comprise a captureprobe 40 attached to an electrode 1 via an attachment linker 45, a labelprobe 55 comprising one or more ETMs 35, and a ligated strand 6comprising ligation probes 20 and 21.

In other embodiments, such as the embodiment illustrated in FIG. 8B, theassay complex can comprise a ligation probe 20 comprising aninterrogation position 15 attached to an electrode 1 via an attachmentlinker 45 to which is added a target strand 5 having a detectionposition 15. A second ligation probe 21 with an ETM 35 can then beadded. If the base at the interrogation position is complementary to thebase present at the detection position, a ligated strand will be formedthat can be detected following disassociation of the target strand.

In yet other embodiments, such as illustrated in FIG. 8C, the assaycomplex can comprise a ligated strand 6 and a label probe 55 comprisinga first portion that hybridizes to the ligated strand 6 and a secondportion comprising a recruitment linker 75 and ETMs 35.

The assays are generally run under stringency conditions which allowsformation of an assay complex only in the presence of a substantiallycomplementary capture probe. Alternately, the hybridization complex cancomprise the target sequence, a two ligation probes, one of which isattached to an electrode via an attachment linker. Stringency can becontrolled by altering a step parameter that is a thermodynamicvariable, including, but not limited to, temperature, formamideconcentration, salt concentration, chaotropic salt concentration pH,organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions; for example,when an initial hybridization step is done between the ligated strandand the capture probe. Running this step at conditions which favorspecific binding can allow the reduction of non-specific binding.

When all of the components outlined herein are used, a preferred methodis as follows. The ligated strand is disassociated from the targetstrand using heat denaturation and incubated under hybridizationconditions with a capture probe and a label probe. Generally, thisreaction is carried out in the presence of an electrode with at leastone immobilized capture probe, although this may also be done in twosteps, with the initial incubation and the subsequent addition to theelectrode. An optional wash step can be used to remove excess reagents,and detection of the ligated strand proceeds as outlined below.

In one embodiment, a number of capture probes (or capture probes andcapture extender probes) that are each substantially complementary to adifferent portion of the target sequence are used.

As will be appreciated by persons skilled in the art, other methods fordetecting the ligated strand are available. For example, other methodscan utilize a ligation probe attached to an electrode to which is addeda second ligation probe and a target strand. The ligated strand caneither be detected directly following disassociation of the targetstrand, or indirectly following addition of a capture probe comprising arecruitment linker.

The ligated strand can be detected in a variety of ways. In a preferredembodiment, one of the probes comprises at least one covalently attachedETM, and the other probe comprises a sequence that is used to hybridizeeither directly or indirectly (i.e. through the use of a captureextender probe) to a capture probe on an electrode. For example, thecapture probe can hybridize to the second ligation probe (FIG. 8A) or toa first portion of a capture extender probe. The capture extender probecomprises a first portion that hybridizes to the capture probe and asecond portion that hybridizes to the second ligation probe (see, e.g.,FIG. 14 in WO 00/20476 for a description of capture extender probes; thedisclosure of which is incorporated herein by reference in itsentirety). Only if both components are present will a signal begenerated; this can eliminate the need for removing unligated probesfrom the system. Alternatively, unligated probes can be removed orwashed away, for example using a binding step, etc.

Alternatively, rather than have the probe directly labeled with an ETM,sandwich assay systems can be used (see WO 00/20476 for a description ofsandwich assays, the disclosure of which is incorporated herein byreference in its entirety). For example, as shown in FIG. 8A, a sandwichassay comprising capture probe 40 with a sequence complementary to theportion of a ligated strand comprising ligation probe 20 and a labelprobe 55 comprising at least one ETM and having a sequence complementaryto the portion of the ligated strand comprising ligation probe 21 can beused to detect the ligated strand. Other embodiments utilize amplifierprobes, label extender probes, etc. as described in WO 00/20476.

If, as illustrated in FIG. 8A, the ligation reaction is done insolution, the ligation complex can be denatured and the ligated strandadded to a detection electrode. Preferred embodiments utilize theseparation of the ETM label and the capture sequences on differentprobes. This minimizes or prevents unligated probes comprising ETMs frombeing captured on the surface. Alternatively, the ligation reaction canbe done on a surface, with the capture of the target sequence and thenthe recruitment of the probe comprising the label (or a probe to which alabel probe will bind) to the target sequence (FIG. 8B). Generally, thisembodiment utilizes a thermal step to drive off unligated probes, suchthat only the ligated strands remain on the surface. Similarly, thecapture probe itself can be used as a ligation probe, with its terminuscomprising the detection position (FIG. 8C). Upon addition of the targetsequence and a second ligation probe, a ligation complex can be formed.A label probe (or other probes) can be added as well. Again, thisembodiment may require the use of a thermal step to ensure that thetarget sequence does not remain on the surface unless ligation hasoccurred.

Accordingly, in a preferred embodiment, the present invention providesarrays, each array location comprising at a minimum a covalentlyattached nucleic acid probe, such as a capture probe or a ligationprobe. By “array locations” or “pads” or “sites” herein meant a locationon the substrate that comprises a covalently attached nucleic acidprobe. The array locations may comprise electrodes and self-assembledmonolayers (SAMs). By “array” herein is meant a plurality of nucleicacid probes in an array format; the size of the array will depend on thecomposition and end use of the array. Arrays containing from about 2different capture ligands to many thousands can be made. Generally, thearray will comprise from two to as many as 100,000 or more, depending onthe size of the electrodes, as well as the end use of the array.Preferred ranges are from about 2 to about 10,000, with from about 5 toabout 1000 being preferred, and from about 10 to about 100 beingparticularly preferred. In some embodiments, the compositions of theinvention may not be in array format; that is, for some embodiments,compositions comprising a single capture ligand may be made as well. Inaddition, in some arrays, multiple substrates may be used, either ofdifferent or identical compositions. Thus, for example, large arrays maycomprise a plurality of smaller substrates.

In a preferred embodiment, the arrays are present on a substrate. By“substrate” or “solid support” or other grammatical equivalents hereinis meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association ofnucleic acids. The substrate can comprise a wide variety of materials,as will be appreciated by those in the art, with printed circuit board(PCB) materials being particularly preferred. A description of printedcircuit board materials can be found in WO 00/20476 and U.S. Ser. Nos.09/993,342 and 10,412,660. For a description of other suitablesubstrates that may be used in the methods and compositions of thepresent invention, as well as descriptions of the arrays and methods ofmaking them, see WO 00/20476 and U.S. Ser. Nos. 09/993,342 and10,412,660, the disclosures of which are incorporated herein byreference.

The substrates can comprise electrodes. By “electrode” herein is meant acomposition, which, when connected to an electronic device, is able tosense a current or a potential and convert it to a signal. Alternativelyan electrode can be defined as a composition which can apply a potentialto and/or pass electrons to or from species in the solution. Thus, anelectrode is an ETM as described below. Preferred electrodes are knownin the art and include, but are not limited to, certain metals and theiroxides, including gold; platinum; palladium; silicon; aluminum; metaloxide electrodes including platinum oxide, titanium oxide, tin oxide,indium tin oxide, palladium oxide, silicon oxide, aluminum oxide,molybdenum oxide (Mo₂O₆), tungsten oxide (WO₃) and ruthenium oxides; andcarbon (including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, platinum, carbon and metaloxide electrodes, with gold being particularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs and probes bound to theinner surface. Electrode coils or mesh may be preferred in someembodiments as well. This allows a maximum of surface area containingthe nucleic acids to be exposed to a small volume of sample.

In addition, the detection electrode may be configured to maximize thecontact the entire sample has with the electrode, or to allow mixing,etc. See for example WO 00/20476 and U.S. Ser. Nos. 09/993,342 and10,412,660; the disclosures of which are incorporated herein byreference in their entirety.

Accordingly, in a preferred embodiment, the present invention providesbiochips (sometimes referred to herein “chips”) that comprise substratescomprising a plurality of electrodes, preferably gold electrodes. Thenumber of electrodes is as outlined for arrays. Each electrodepreferably comprises a self-assembled monolayer (SAM) comprising one ormore species (i.e. mixed SAM), including, but not limited to conductiveoligomers and/or insulators. See U.S. Publication Nos. 2002/0177135 and20020121314 for monolayer forming species, methods of making anddepositing SAMS on solid substrates, and compositions and methods usingSAMs; the disclosures of which are incorporated herein by reference.

In a preferred embodiment, one of the monolayer-forming speciescomprises a capture probe as described in WO 00/20476, the disclosure ofwhich is incorporated herein by reference in its entirety. In addition,each electrode has an interconnection, that is attached to the electrodeat one end and is ultimately attached to a device that can control theelectrode. That is, each electrode is independently addressable (seeU.S. Publication No. 2002/0177135 and U.S. Ser. No. 10,412,660; thedisclosures of which are incorporated herein by reference).

The substrates can be part of a larger device comprising a detectionchamber that exposes a given volume of sample to the detectionelectrode. Generally, the detection chamber ranges from about 1 nL to 1ml, with about 10 μL to 500 μL being preferred. As will be appreciatedby those in the art, depending on the experimental conditions and assay,smaller or larger volumes may be used. See also U.S. Ser. No.09/295,691, incorporated herein by reference in its entirety.

In some embodiments, the detection chamber and electrode are part of acartridge that can be placed into a device comprising electroniccomponents (an AC/DC voltage source, an ammeter, a processor, a read-outdisplay, temperature controller, light source, etc.). In thisembodiment, the interconnections from each electrode are positioned suchthat upon insertion of the cartridge into the device, connectionsbetween the electrodes and the electronic components are established(see U.S. Publication No. 2002/0177135 and U.S. Serial No. 10,412,660;the disclosures of which are incorporated herein by reference).

Preferably, one of three basic detection mechanisms can be used todetect the ligated strand. In a preferred embodiment, detection relieson the presence of one or more ETMs. Detection of the ETM is based onelectron transfer through the stacked π-orbitals of double strandednucleic acid. This basic mechanism is described in U.S. Pat. Nos.5,591,578, 5,770,369, 5,705,348, and PCT US97/20014 and is termed“mechanism-1”. Briefly, previous work has shown that electron transfercan proceed rapidly through the stacked π-orbitals of double strandednucleic acid, and significantly more slowly through single-strandednucleic acid. Accordingly, this can serve as the basis of an assay.Thus, by adding ETMs (either covalently to one of the strands ornon-covalently to the assay complex through the use of hybridizationindicators, see U.S. Pat. No. 5,952,172 for a description ofhybridization indicators or mediators, the disclosure of which isincorporated herein by reference in its entirety) to a nucleic acid thatis attached to a detection electrode via a conductive oligomer, electrontransfer between the ETM and the electrode, through the nucleic acid andconductive oligomer, may be detected. See also U.S. Pat. Nos. 6,096,273,6,221,583, and 6,090,933; the disclosures of which are incorporatedherein by reference.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected; thatis, the electrons from the ETMs need not travel through the stacked πorbitals in order to generate a signal. This basic idea is termed“mechanism-2”. In this embodiment, the detection electrode preferablycomprises a self-assembled monolayer (SAM) that serves to shield theelectrode from redox-active species in the sample. The SAM can beformulated to comprise slight “defects” (sometimes referred to herein as“microconduits”, “nanoconduits” or “electroconduits”). Essentially, theelectroconduits allow particular ETMs access to the surface. Withoutbeing bound by theory, it should be noted that the configuration of theelectroconduit depends in part on the ETM chosen. For example, the useof relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs. See also, U.S.Publication No. 2002/0177135 and U.S. Serial No. 10,412,660 for ageneral discussion of electroconduits and methods of making; thedisclosures of which are incorporated herein by reference.

Finally, reporterless or labelless systems can also be used to detectSNPs. In these systems, two detection electrodes are used to measurechanges in capacitance or impedance as a result of target analytebinding. See generally U.S. Ser. No. 09/458,533, filed Dec. 9, 1999 andPCT US00/33497, the disclosures of which are incorporated herein byreference.

In a preferred embodiment, the detection electrode further comprises acapture probe, preferably covalently attached. By “capture probe” hereinis meant a nucleic acid sequence that is used to probe for the presenceof the ligated strand, and that binds the ligated strand in a sequencespecific manner. In general, for most of the embodiments describedherein, at least one capture probe is used per ligated strand.

Generally, the capture probe allows the attachment of the ligated strandor a ligation probe to the detection electrode, for the purposes ofdetection. Attachment of the ligated stand to the capture probe may bedirect (i.e. the ligated probe binds to the capture probe) or indirect(one or more capture extender probes may be used).

As will be appreciated by those in the art, the composition of thecapture probe will depend on the composition of the ligated strand orligation probe. For example, when the ligated strand is asingle-stranded nucleic acid, the capture probe is generally asubstantially complementary nucleic acid.

Preferred compositions and techniques are outlined in WO 98/20162;PCT/US98/12430; PCT/US98/12082; PCT/US99/01705; PCT/US99/01703; and U.S.Ser. Nos. 09/135,183; 60/105,875; and 09/295,691, for nucleic acidcapture probes, all of which are hereby expressly incorporated byreference.

The capture probe can be attached to the electrode via an attachmentlinker, such as an insulator or conductive oligomer. See WO 00/20476,U.S. Publication No. 20020121314, U.S. Pat. Nos. 6,096,273, 6,221,583,and 6,090,933 for a discussion of means of attaching capture probes toan electrode vian an attachment linker; the disclosures of which areincorporated herein by reference. Other linkers can also be used; forexample, homo-or hetero-bifunctional linkers (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). In general, the capture probes areattached to the attachment linker through the use of functional groupson each that can then be used for attachment. Preferred functionalgroups for attachment are amino groups, carboxy groups, oxo groups andthiol groups.

In a preferred embodiment, there may be one or more different captureprobe species on the surface. In some embodiments, there may be one typeof capture probe, or one type of capture probe extender. “Captureextender” probes are generally depicted in FIG. 14 of WO 00/20476, andhave a first portion that will hybridize to all or part of the captureprobe, and a second portion that will hybridize to a portion of ligatedstrand. Alternatively, different capture probes, or one capture probeswith a multiplicity of different capture extender probes can be used.Similarly, it may be desirable (particular in the case of nucleic acidtarget sequences in mechanism-2 systems) to use auxiliary capture probesthat comprise relatively short probe sequences, that can be used to“tack down” components of the system, for example the recruitmentlinkers, to increase the concentration of ETMs at the surface. See, WO00/20476, for a description of these embodiments.

In a preferred embodiment, the assay complexes further comprise a label,solution or soluble binding probe, although, for mechanism-1 systems,the ETMs may be added in the form of non-covalently attachedhybridization indicators. Solution binding probes are similar to captureprobes, in that they bind, preferably specifically, to specificsequences present on the ligated strand. The solution binding probe maybe the same or different from the capture probe. Generally, the solutionbinding probes are not directed attached to the surface, although theymay be. The solution binding probe either directly comprises arecruitment linker that comprises at least one ETM, or the recruitmentlinker binds, either directly or indirectly, to the solution bindingprobe.

Thus, “solution binding probes” or “soluble binding probes” or “signalcarriers” or “label probes” or “label binding probes” with recruitmentlinkers comprising covalently attached ETMs are provided. That is, oneportion of the label probe or solution binding probe directly orindirectly binds to the ligated strand, and one portion comprises arecruitment linker comprising covalently attached ETMs. In some systems,for example in mechanism-1 nucleic acid systems, these may be the same.

The compositions and methods of the invention find use in a variety ofapplications. In a preferred embodiment, the present invention finds usein detecting target sequences by detecting ligated strands resultingfrom the chemical ligation of two ligation probes. For example, geneexpression analyses may be done, or straight detection of the presenceor absence of target sequences. For example, straight detection can beused to detect pathogens and for forensic analysis. In this embodiment,a ligation substrate is formed comprising a target sequence, a firstligation probe with an iodide moiety attached at the 5′ end that iscomplementary to a first domain on the target sequence and a secondligation probe with at least one ETM and sulfur moiety attached at the3′ end, complementary to a second domain of the target sequence. Thatis, the target sequence acts as a “catalyst” orienting the ligationprobes in the correct orientation for chemical ligation to occur. Ifthere is no mismatch between the first and second ligation probes andthe target sequence, chemical ligation occurs, in which a covalentlinkage is formed when the 5′-iodide moiety on the first ligation probeis displaced by the 3′-sulfur moiety on the second ligation. The ligatedstrand may then be separated from the target sequence via denaturationand hybridized to a capture probe on an electrode for electrochemicaldetection as described herein.

It should be noted in this context that “mismatch” is a relative termand meant to indicate a difference in the identity of a base at aparticular position, termed the “detection position” herein, between twosequences. In general, sequences that differ from wild type sequencesare referred to as mismatches. However, particularly in the case ofSNPs, what constitutes “wild type” may be difficult to determine asmultiple alleles can be relatively frequently observed in thepopulation, and thus “mismatch” in this context requires the artificialadoption of one sequence as a standard. Thus, for the purposes of thisinvention, sequences are referred to herein as “perfect match” and“mismatch”.

In a preferred embodiment, the present invention finds use in SNPdetection and discovery. By “SNP” or “single nucleotide polymorphism”herein is meant a difference or variation, i.e. polymorphism in a singlenucleotide. As will be appreciated by those of skill in the art, a SNPmay comprise a plurality of nucleotides, either contiguous with eachother or separated by one or more nucleotides.

In a preferred embodiment, SNPs are detected using multiple ligationprobes (also referred to herein as a ligation probe set). Preferably,each ligation probe set has a different base at the interrogationposition and a different covalently attached ETM with a different redoxpotential. Thus, sets of two probes (for example, when a SNP may existas one of two different bases), three probes (when an allele comprises 3different bases) or four probes (to determine the identity of the baseat the detection position) can be used. By adding the set of probes tothe target sequence and detecting which ETM is present, the identity ofthe base at the detection position is determined.

In a preferred embodiment, all of the other positions of the probes usedin this embodiment are the same; that is, in some embodiments it ispreferable to use probes that have all other components equal (e.g. boththe length of the probes as well as the non-interrogation bases) toallow good discrimination. This is particularly preferred for SNPdiscovery and analysis.

Once the assay complexes of the invention are made, the presence of theETMs at the surface of the monolayer can be detected in a variety ofways. A variety of detection methods may be used, including, but notlimited to, optical detection (as a result of spectral changes uponchanges in redox states), which includes fluorescence, phosphorescence,luminiscence, chemiluminescence, electrochemiluminescence, andrefractive index; and electronic detection, including, but not limitedto, amperommetry, voltammetry, capacitance and impedence. These methodsinclude time or frequency dependent methods based on AC or DC currents,pulsed methods, lock-in techniques, filtering (high pass, low pass, bandpass), and time-resolved techniques including time-resolvedfluorescence.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred.Without being limited by the mechanism or theory, detection is based onthe transfer of electrons from the ETM to the electrode. Methods ofdetecting electron transfer are described in WO 00/20476, WO 00/16089and U.S. Publication Nos. 20030143556, 20020177135, 20020121314, thedisclosures of which are hereby incorporated by reference.

In some embodiments, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

Alternative electron detection modes also can be utilized. For example,potentiometric (or voltammetric) measurements involve non-faradaic (nonet current flow) processes and are utilized traditionally in pH andother ion detectors. Similar sensors are used to monitor electrontransfer between the ETM and the electrode. In addition, otherproperties of insulators (such as resistance) and of conductors (such asconductivity, impedance and capacitance) could be used to monitorelectron transfer between ETM and the electrode. Finally, any systemthat generates a current (such as electron transfer) also generates asmall magnetic field, which may be monitored in some embodiments.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents. See WO 00/20476, WO 00/16089 and U.S. Publication Nos.20030143556, 20020177135, 20020121314, the disclosures of which arehereby incorporated by reference.

In a preferred embodiment, AC initiation and detection methods are used.When AC initiation and detection methods are used, the frequencyresponse of the system changes as a result of the presence of the ETM.By “frequency response” herein is meant a modification of signals as aresult of electron transfer between the electrode and the ETM. Thismodification is different depending on signal frequency. A frequencyresponse includes AC currents at one or more frequencies, phase shifts,DC offset voltages, faradaic impedance, etc. See WO 00/20476, WO00/16089 and U.S. Publication Nos. 20030143556, 20020177135, 20020121314for a discussion of AC initiation and detection methods.

The use of combinations of AC and DC initiation signals gives a varietyof advantages, including surprising sensitivity and signal maximization.Accordingly, in a preferred embodiment, the first input signal comprisesa DC component and an AC component. That is, a DC offset voltage betweenthe sample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM. See WO 00/20476, WO 00/16089 and U.S. Publication Nos.20030143556, 20020177135, 20020121314 for a discussion of AC and DCinitiation and detection methods.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Non-Enzymatic Ligation

The efficiency of chemical ligation using phosphate-amine chemicalligation chemistry was analyzed. The ligation assay consisted of: 1)target DNA with two matched probes; and, 2) target DNA with onemismatch.

As shown in FIG. 9, the reaction involves a two step process by which1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) first binds to aligation probe labeled with a 3′-phosphate, followed by nucleophillicattack on a 5′-amino group present on a second ligation probe, to form aphosphoramidate bond. The 5′-amino ligation probe was made using GlenResarch's 5′-amino-dT phosphoramidite. The ligation probe comprising the3′-phosphate was made using H8 (see FIG. 10).

HPLC analysis was used to detect the peak eluting at 20.3 minutes thatcorresponded to the ligated strand. In the presence of a complementarytarget strand, greater than 90% ligation efficiency was observed inapproximately 4 hours at 16° C. (FIG. 11A). Greater that 48% efficiencywas observed at 2 hours (FIG. 11B) and greater than 95% efficiency wasobserved with an overnight incubation (FIG. 11C). In contrast, no peakwas observed in a complex containing a mismatch following overnightincubation (FIG. 11D).

Surface hybridization and electrochemical analysis were preformed toassess the sensitivity of the electrochemical assay to distinguishbetween “match” and “mismatch”. Samples were taken from thehybridization cocktail at different times and measured electronically.As shown in FIG. 12, the electrochemical assay was capable ofdistinguishing between a matched complex and a mismatched complex.

1. A composition comprising: a) a first ligation probe comprising a 5′-iodide moiety; and b) a second ligation comprising a 3′-sulfur moiety; wherein at least one of said ligation probes comprises at least a first electron transfer moiety with a first redox potential.
 2. A composition according to claim 1 further comprising a target nucleic acid strand with a first domain that hybridizes to said first ligation probe and a second domain that hybridizes to said second ligation probe.
 3. A composition according to claim 1 further comprising a third ligation probe comprising a second ETM with a second redox potential different from said first redox potential.
 4. A composition according to claim 1 further comprising a fourth ligation probe comprising a third ETM with a third redox potential different from said first or said second redox potential.
 5. A composition according to claim 1 further comprising a fifth ligation probe comprising a fourth ETM with a fourth redox potential different from said first, said second or said third redox potential.
 6. A composition according to claim 3 wherein said first ligation probe comprises an interrogation position with a first base and said third ligation probe comprises an interrogation position with a second base.
 7. A composition according to 5 wherein said first ligation probe comprises an interrogation position with a first base, said third ligation probe comprises an interrogation position with a second base, said fourth ligation probe comprises an interrogation position with a third base, and said fifth ligation probe comprises an interrogation position with a fourth base.
 8. A composition according to claim 7 wherein said first, third, fourth and fifth ligation probe comprise the 5′ iodide moiety.
 9. A composition according to claim 7 wherein said first, third, fourth and fifth ligation probe comprise the 3′ sulfur moiety.
 10. A method of chemical ligation, said method comprising: a) providing a ligation substrate comprising: i) a target nucleic acid strand; ii) a first ligation probe comprising a 5′-iodide moiety; and iii) a second ligation probe comprising a 3′ sulfur moiety; wherein at least one of said first and said second ligation probes comprises at least a first electron transfer moiety with a first redox potential; b) ligating said first and said second ligation probes under conditions wherein said 5′-iodide moiety on said first ligation probe is displaced by said 3′-sulfur moiety on said second ligation probe to form a covalent linkage.
 11. A method of detecting a target sequence in a sample comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first domain and an adjacent second domain; ii) a first ligation probe that is hybridized to said first domain, wherein said first ligation probe comprises a 5′-iodide moiety; iii) a second ligation probe that is hybridized to said second domain, wherein said second ligation probe comprises a 3′-sulfur moiety; wherein at least one of said first and second ligation probes comprise at least one electron transfer moiety (ETM); b) forming a ligation complex comprising said target sequence and a ligated strand by ligating said first and said second ligation probes under conditions wherein said 5′-iodide moiety on said first ligation probe is displaced by said 3′-sulfur moiety on said second ligation probe to form a ligated strand; c) denaturing said ligation complex; d) hybridizing said ligated strand to a capture probe on an electrode; and e) detecting said electron transfer moiety as an indication of the presence of said target sequence in said sample.
 12. A method according to claim 11 wherein said first ligation probe comprises said 3′-sulfur moiety and said second ligation probe comprises said 5′-iodide moiety.
 13. A method according to claim 11 wherein said ETM is a ferrocene.
 14. A method according to claim 11 wherein at least one of said ligation probes comprises a recruitment linker comprising two or more ETMs.
 15. A method according to claim 11 wherein said electrode further comprises a self-assembled monolayer.
 16. A method of detecting a target sequence in a sample comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first domain and an adjacent second domain; ii) an electrode comprising a first ligation probe covalently attached to said electrode via an attachment linker, wherein said first ligation probe is hybridized to said first domain of said target sequence; and, iii) a second ligation probe comprising at least one ETM and wherein said second ligation probe is hybridized to said second domain of said target sequence; wherein said first ligation probe comprises a 3′-sulfur moiety and said second ligation probe comprises a 5′-iodide moiety; b) forming a ligation complex comprising said target sequence and a ligated strand by ligating said first and said second ligation probes under conditions wherein said 5′-iodide moiety on said first ligation probe is displaced by said 3′-sulfur moiety on said second ligation probe to form a ligated strand; c) denaturing said ligation complex, such that said ligated strand comprising said first and said second ligation probes is attached to said electrode; and d) detecting said electron transfer moiety as an indication of the presence of said target sequence in said sample.
 17. A method of detecting a target sequence in a sample comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first domain and an adjacent second domain; ii) an electrode comprising a first ligation probe covalently attached to said electrode via an attachment linker, wherein said first ligation probe is hybridized to said first domain of said target sequence; and, iii) a second ligation probe hybridized to said second domain of said target sequence; wherein said first ligation probe comprises a 3′-sulfur moiety and said second ligation probe comprises a 5′-iodide moiety; b) forming a ligation complex comprising said target sequence and a ligated strand by ligating said first and said second ligation probes under conditions wherein said 5′-iodide moiety on said first ligation probe is displaced by said 3′-sulfur moiety on said second ligation probe to form a ligated strand; c) denaturing said ligation complex, such that said ligated strand comprising said first and said second ligation probe is covalently attached to said electrode via said attachment linker; d) hybridizing said ligated strand to a label probe comprising at least one ETM; and e) detecting said electron transfer moiety as an indication of the presence of said target sequence in said sample.
 18. A method according to claim 16 or 17 wherein said first ligation probe comprises said 5′-idodide moiety and said second ligation probe comprises said 3′-sulfur moiety moiety.
 19. A method according to claim 16 or 17 wherein said ETM is a ferrocene.
 20. A method according to claim 17 wherein said label probe comprises a recruitment linker comprising two or more ETMs.
 21. A method according to claim 16 or 17 wherein said electrode further comprises a self-assembled monolayer.
 22. A method of detecting a target sequence in a sample comprising: a) providing a substrate comprising an array of electrodes each comprising: i) a self-assembled monolayer (SAM); and, ii) a different first ligation probe, wherein each of said first ligation probes is capable of hybridizing to a first domain of a target sequence: iii) a plurality of target sequences comprising a first domain and an adjacent second domain; iv) a plurality of second ligation probes capable of hybridizing to said second domain of said target sequences; wherein said first ligation probes comprise a 3′-sulfur moiety and said second ligation probes comprise a 5′-iodide moiety; b) forming ligation complexes comprising said target sequences and a plurality of ligated strands by ligating said first and said second ligation probes under conditions wherein said 5′-iodide moiety on said first ligation probes displaces said 3′-sulfur moiety on said second ligation probes to form a plurality of ligated strands; c) denaturing said ligation complexes, such that said ligated strands are covalently attached to said electrodes; d) hybridizing said ligated strands to a plurality of label probes comprising at least one ETM; and e) detecting said electron transfer moiety as an indication of the presence of said target sequences in said sample.
 23. A method for ligating two probes comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first domain and an adjacent second domain; ii) a first ligation probe comprising a 3′ OH moiety; iii) a second ligation probe comprising a 5′ OH moiety; wherein at least one of said ligation probes comprises at least one covalently attached electron transfer moiety; b) contacting said ligation substrate with a ligation chemical to ligate said ligation probes to form a ligated strand.
 24. A method according to claim 23 wherein said ligation chemical is N-cyanoimidazole.
 25. A method according to claim 23 wherein said ligation chemical is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
 26. A method for ligating two probes comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first domain and an adjacent second domain; ii) a first ligation probe comprising a 3′ phosphate moiety; iii) a second ligation probe comprising a 5′ amino moiety; wherein at least one of said ligation probes comprises at least one covalently attached electron transfer moiety; b) contacting said ligation substrate with a ligation chemical to ligate said ligation probes to form a ligated strand.
 27. A method according to claim 26 wherein said ligation chemical is N-cyanoimidazole.
 28. A method according to claim 26 wherein said ligation chemical is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
 29. A method for determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, said method comprising: a) hybridizing a first ligation probe to said first domain, wherein said first ligation probe comprises: i) a first base at an interrogation position; ii) a 3′-sulfur moiety; and iii) a first electron transfer moiety comprising a first redox potential; b) hybridizing a second ligation probe to said second domain, wherein said second probe comprises a 5′-iodide moiety; wherein if said first base of said first ligation probe is perfectly complementary to said detection position a ligation substrate is formed; c) providing conditions wherein said 3′-sulfur moiety on said first ligation probe displaces said 5′-iodide moiety on said second ligation probe to form a ligated strand; d) forming an assay complex comprising said ligated strand and a capture probe covalently attached to an electrode; e) detecting the presence or absence of said ETM as an indication of the formation of said ligated strand; and f) identifying the base at said detection position.
 30. A method for determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain and a second target domain adjacent to said first domain and comprising said detection position, said method comprising: a) hybridizing a first ligation probe to said first domain, wherein said first ligation probe comprises: i) a first base at an interrogation position; ii) a 5′-iodide moiety; and iii) a first electron transfer moiety comprising a first redox potential; b) hybridizing a second ligation probe to said second domain, wherein said second probe comprises a 3′-sulfur moiety; wherein if said first base of said first ligation probe is perfectly complementary to said detection position a ligation substrate is formed; c) providing conditions wherein said 3′-sulfur moiety on said second ligation probe displaces said 5′-iodide moiety on said first ligation probe to form a ligated strand; d) forming an assay complex comprising said ligated strand and a capture probe covalently attached to an electrode; e) detecting the presence or absence of said ETM as an indication of the formation of said ligated strand; and f) identifying the base at said detection position. 